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PCR产物纯化方法 Purification of PCR Products in Preparation for Cloning
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PurificationofPCRProductsinPreparationforCloning JosephSambrookPeterMaccallumCancerInstituteandTheUniversityofMelbourne,AustraliaDavidW.RussellUniversityofTexasSouthwesternMedicalCenter,DallasExcerptedFromMolecularCloning:ALaboratoryManualThirdEdition ABSTRACT TheresidualenzymaticactivityofThermostableDNApolymerasesthatsurvivetherigorsofPCRcancompromisesubsequentenzymaticreactions.ThisprotocoldescribeshowtouseproteinaseKtodestroythermostableenzymesandtopurifyamplifiedDNAinpreparationforcloning. MATERIALS ReagentsandSolutions EnzymesandBuffers Gels METHOD TROUBLESHOOTING Nonspecificamplificationproductsarepresentatsignificantlevels(e.g.,aredetectablebygelelectrophoresis).Step1Purifythedesiredproductbyelectrophoresisthroughlow-melting-temperatureagarosebeforeproceeding(pleaseseeRecoveryofDNAfromLow-melting-temperatureAgaroseGels:OrganicExtraction). REFERENCES CroweJ.S.,CooperH.J.,SmithM.A.,SimsM.J.,ParkerD.,GewertD.1991.Improvedcloningefficiencyofpolymerasechainreaction(PCR)productsafterproteinaseKdigestion.NucleicAcidsRes.19:184-184.[FreeFullText]WybranietzW.A.andLauerU.1998.Distinctcombinationofpurificationmethodsdramaticallyimprovescohesive-endsubcloningofPCRproducts.BioTechniques24:578-580.[Medline] Anyoneusingtheproceduresinthisprotocoldoessoattheirownrisk.ColdSpringHarborLaboratorymakesnorepresentationsorwarrantieswithrespecttothematerialsetforthinthisprotocolandhasnoliABIlityinconnectionwiththeuseofthesematerials.Materialsusedinthisprotocolmaybeconsideredhazardousandshouldbeusedwithcaution.Forafulllistingofcautionsregardingthesematerial,pleaseconsult:MolecularCloning:ALaboratoryManualThirdEdition,byJosephSambrookandDavidW.Russell,©2001byColdSpringHarborLaboratoryPress,ColdSpringHarbor,NewYork,p..8.25-8.26. Copyright©2006byColdSpringHarborLaboratoryPress.Allrightsreserved.Nopartofthesepages,eithertextorimagemaybeusedforanypurposeotherthanpersonaluse.Therefore,reproductionmodification,storageinaretrievalsystemorretransmission,inanyformorbyanymeans,electronic,mechanical,orotherwise,forreasonsotherthanpersonaluse,isstrictlyprohibitedwithoutpriorwrittenpermission
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